Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

doi:10.1038/s41589-024-01702-5

. 2024 Nov;20(11):1535-1546.

doi: 10.1038/s41589-024-01702-5. Epub 2024 Aug 13.

Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

Derek Aspacio^#¹, Yulai Zhang^#¹, Youtian Cui^#², Emma Luu², Edward King³, William B Black¹, Sean Perea¹, Qiang Zhu^{1

3

4

5}, Yongxian Wu^{1

3

4

5}, Ray Luo^{1

3

4

5}, Justin B Siegel^{2

6

7}, Han Li^{8

9

10}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA.
² Genome Center, University of California, Davis, Davis, CA, USA.
³ Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA.
⁴ Department of Material Science and Engineering, University of California, Irvine, Irvine, CA, USA.
⁵ Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA.
⁶ Department of Chemistry, University of California, Davis, Davis, CA, USA.
⁷ Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, USA.
⁸ Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA. han.li@uci.edu.
⁹ Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. han.li@uci.edu.
¹⁰ Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA. han.li@uci.edu.

^# Contributed equally.

PMID: 39138383
DOI: 10.1038/s41589-024-01702-5

Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

Derek Aspacio et al. Nat Chem Biol. 2024 Nov.

. 2024 Nov;20(11):1535-1546.

doi: 10.1038/s41589-024-01702-5. Epub 2024 Aug 13.

Authors

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA.
² Genome Center, University of California, Davis, Davis, CA, USA.
³ Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA.
⁴ Department of Material Science and Engineering, University of California, Irvine, Irvine, CA, USA.
⁵ Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA.
⁶ Department of Chemistry, University of California, Davis, Davis, CA, USA.
⁷ Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, USA.
⁸ Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA. han.li@uci.edu.
⁹ Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. han.li@uci.edu.
¹⁰ Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA. han.li@uci.edu.

^# Contributed equally.

PMID: 39138383
DOI: 10.1038/s41589-024-01702-5

Abstract

Nature's two redox cofactors, nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN⁺) as a noncanonical cofactor orthogonal to NAD(P)⁺. Here we present the development of Nox Ortho, a reduced NMN⁺ (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN⁺ ratio together with an NMN⁺-specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~10³-10⁶-fold cofactor specificity switch from NAD(P)⁺ to NMN⁺. We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)'s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H).

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Update of

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor.
Aspacio D, Zhang Y, Cui Y, King E, Black WB, Perea S, Luu E, Siegel JB, Li H. Aspacio D, et al. bioRxiv [Preprint]. 2023 Aug 30:2023.08.29.555398. doi: 10.1101/2023.08.29.555398. bioRxiv. 2023. Update in: Nat Chem Biol. 2024 Aug 13. doi: 10.1038/s41589-024-01702-5. PMID: 37693387 Free PMC article. Updated. Preprint.

Cited by

Orthogonal redox control.
Hümmler LM, Lindner SN. Hümmler LM, et al. Nat Chem Biol. 2024 Nov;20(11):1395-1396. doi: 10.1038/s41589-024-01728-9. Nat Chem Biol. 2024. PMID: 39317846 No abstract available.

References

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1. Liao, J. C., Mi, L., Pontrelli, S. & Luo, S. Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 14, 288–304 (2016). - PubMed - DOI
1. Opgenorth, P. H., Korman, T. P. & Bowie, J. U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat. Commun. 5, 4113 (2014). - PubMed - DOI
1. Cresko, J. et al. Industrial Decarbonization Roadmap (US Department of Energy, 2022).
1. Lee, S. Y. & Kim, H. U. Systems strategies for developing industrial microbial strains. Nat. Biotechnol. 33, 1061–1072 (2015). - PubMed - DOI

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[1] Liu, Y. et al. Biofuels for a sustainable future. Cell 184, 1636–1647 (2021). - PubMed - DOI

[2] Liu, Y. et al. Biofuels for a sustainable future. Cell 184, 1636–1647 (2021). - PubMed - DOI

[3] Liao, J. C., Mi, L., Pontrelli, S. & Luo, S. Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 14, 288–304 (2016). - PubMed - DOI

[4] Liao, J. C., Mi, L., Pontrelli, S. & Luo, S. Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 14, 288–304 (2016). - PubMed - DOI

[5] Opgenorth, P. H., Korman, T. P. & Bowie, J. U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat. Commun. 5, 4113 (2014). - PubMed - DOI

[6] Opgenorth, P. H., Korman, T. P. & Bowie, J. U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat. Commun. 5, 4113 (2014). - PubMed - DOI

[7] Cresko, J. et al. Industrial Decarbonization Roadmap (US Department of Energy, 2022).

[8] Cresko, J. et al. Industrial Decarbonization Roadmap (US Department of Energy, 2022).

[9] Lee, S. Y. & Kim, H. U. Systems strategies for developing industrial microbial strains. Nat. Biotechnol. 33, 1061–1072 (2015). - PubMed - DOI

[10] Lee, S. Y. & Kim, H. U. Systems strategies for developing industrial microbial strains. Nat. Biotechnol. 33, 1061–1072 (2015). - PubMed - DOI

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Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

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Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

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